Structural and Biochemical Analysis of the Furan Aldehyde Reductase YugJ from Bacillus subtilis

NAD(H)/NADP(H)-dependent aldehyde/alcohol oxidoreductase (AAOR) participates in a wide range of physiologically important cellular processes by reducing aldehydes or oxidizing alcohols. Among AAOR substrates, furan aldehyde is highly toxic to microorganisms. To counteract the toxic effect of furan aldehyde, some bacteria have evolved AAOR that converts furan aldehyde into a less toxic alcohol. Based on biochemical and structural analyses, we identified Bacillus subtilis YugJ as an atypical AAOR that reduces furan aldehyde. YugJ displayed high substrate specificity toward 5-hydroxymethylfurfural (HMF), a furan aldehyde, in an NADPH- and Ni2+-dependent manner. YugJ folds into a two-domain structure consisting of a Rossmann-like domain and an α-helical domain. YugJ interacts with NADP and Ni2+ using the interdomain cleft of YugJ. A comparative analysis of three YugJ structures indicated that NADP(H) binding plays a key role in modulating the interdomain dynamics of YugJ. Noticeably, a nitrate ion was found in proximity to the nicotinamide ring of NADP in the YugJ structure, and the HMF-reducing activity of YugJ was inhibited by nitrate, providing insights into the substrate-binding mode of YugJ. These findings contribute to the characterization of the YugJ-mediated furan aldehyde reduction mechanism and to the rational design of improved furan aldehyde reductases for the biofuel industry.


Aldehyde Reductase Activity of YugJ
As the first step to investigate the catalytic characteristics of YugJ, we analyzed the substrate specificity of YugJ. The reductase activity of recombinant YugJ protein was determined using an array of aldehydes as a putative substrate in the presence of NADPH as a reducing cofactor. YugJ exhibited the highest reductase activity for HMF, in which an aldehyde group is attached to the hydroxymethylated furan ring (Figure 1a). YugJ was also catalytically active for furfural but had lower activity than for HMF. Although YugJ actively catalyzed HMF reduction, YugJ did not drive the reverse reaction. Essentially no enzymatic activity was detected with 2,5-bis(hydroxymethyl)furan, an alcohol counterpart of HMF, in the presence of NADP (Supplementary Figure S1). These observations indicate that YugJ functions as a furan aldehyde reductase rather than as an alcohol dehydrogenase.
In addition to its activity against cyclic aldehydes, YugJ reduced linear aldehydes. Among butyraldehyde, propionaldehyde, and acetaldehyde, YugJ displayed the highest activity for butyraldehyde, a linear four-carbon aldehyde, at a level comparable to that for HMF. In contrast to butyraldehyde, low activity was observed for isobutyraldehyde, a branched four-carbon aldehyde. This substrate length and shape dependence of YugJ activity has also been reported for other aldehyde reductases, such as E. coli YqhD, a close homolog of YugJ [16]. As observed for YugJ, YqhD showed high specificity for butyraldehyde among linear aldehydes. However, YqhD differs from YugJ in furan aldehyde reduction activity. YugJ displayed substantial activities for HMF and furfural, whereas extremely low activity was observed for YqhD [37]. Another difference in substrate speci-ficity was identified for α-oxoaldehydes, including glyoxal and methylglyoxal, which are generated by oxidative stress and cause cellular damage [5]. E. coli employs YqhD to evade the toxic effects of glyoxal and methylglyoxal [17]. In contrast, YugJ displayed negligible activity toward glyoxal and methylglyoxal. In summary, YugJ displays a unique substrate specificity for HMF, suggesting that YugJ contributes to cellular tolerance for furan aldehydes. In addition to its activity against cyclic aldehydes, YugJ reduced linear aldehydes. Among butyraldehyde, propionaldehyde, and acetaldehyde, YugJ displayed the highest activity for butyraldehyde, a linear four-carbon aldehyde, at a level comparable to that for HMF. In contrast to butyraldehyde, low activity was observed for isobutyraldehyde, a branched four-carbon aldehyde. This substrate length and shape dependence of YugJ activity has also been reported for other aldehyde reductases, such as E. coli YqhD, a close homolog of YugJ [16]. As observed for YugJ, YqhD showed high specificity for butyraldehyde among linear aldehydes. However, YqhD differs from YugJ in furan aldehyde reduction activity. YugJ displayed substantial activities for HMF and furfural, whereas extremely low activity was observed for YqhD [37]. Another difference in substrate specificity was identified for α-oxoaldehydes, including glyoxal and methylglyoxal, which are generated by oxidative stress and cause cellular damage [5]. E. coli employs YqhD to evade the toxic effects of glyoxal and methylglyoxal [17]. In contrast, YugJ displayed negligible activity toward glyoxal and methylglyoxal. In summary, YugJ displays a unique substrate specificity for HMF, suggesting that YugJ contributes to cellular tolerance for furan aldehydes.  To further explore the enzymatic properties of YugJ, the pH dependence of YugJ was examined in a range of pH 6.0-9.5. The catalytic activity of YugJ at pH values below 6.0 could not be addressed due to NADPH instability and the inability of YugJ to bind metals at low pH [38]. YugJ protein exhibited higher HMF-reducing activity at pH 6.0-7.0 than at alkaline pH values (Figure 1b). In addition to pH dependence, YugJ displayed metal dependence. When YugJ protein was demetallized using EDTA at pH 5.0, the metal-free YugJ protein did not reduce HMF at pH 7.4 ( Figure 1c). However, when supplemented with divalent metal ions, YugJ reduced HMF. The highest catalytic activity of YugJ was observed with Ni 2+ and Co 2+ , followed by Zn 2+ , Fe 2+ , Ca 2+ , Mn 2+ , Mg 2+ , and Cu 2+ . In general, Ni 2+ and Co 2+ ions exhibit similar coordination properties, and many Ni 2+ -coordinating proteins can use Co 2+ as an alternative metallic cofactor [39,40]. Therefore, YugJ is expected to mediate the catalytic reaction with Co 2+ in a manner similar to that observed with Ni 2+ . To evaluate the temperature dependence of YugJ, HMF reduction activity was measured at different temperatures ( Figure 1d). The HMF reduction activity of YugJ gradually increased with increasing temperature between 16 and 44 • C and rapidly decreased above 44 • C. Negligible activity was observed at 58 • C. Furthermore, the kinetic parameters of YugJ were derived by fitting the Michaelis-Menten model to the catalytic activities of YugJ that were obtained with a wide range of HMF concentrations in the presence of NADPH and Ni 2+ at pH 7.4, which corresponds to the pH value of the B. subtilis cytosol (Figure 1e) [41]. YugJ showed high catalytic efficiency (k cat /K m , 665 ± 74 M −1 sec −1 ) with a k cat value of 2.55 ± 0.08 sec −1 and a K m value of 3.88 ± 0.53 mM.

Metal Ion Recognition by YugJ
To obtain structural insights into YugJ-mediated aldehyde reduction, we determined the crystal structure of YugJ in complex with Ni 2+ ions (YugJ Ni ) at a 2.15 Å resolution in space group P2 1 2 1 2 1 by molecular replacement (Figure 2a and Table 1). The asymmetric unit of the YugJ crystal contains two YugJ polypeptide chains (chains A and B), which are arranged into a homodimer (Figure 2b). Such a dimer was also detected in solution. In gel-filtration chromatography, YugJ protein (calculated molecular weight of a monomer, 43.3 kDa) was eluted as a dimer (apparent molecular weight,~74 kDa) between 44 and 158 kDa protein standards (Supplementary Figure S2).  YugJ Ni consists of two domains, an N-terminal domain (NTD; residues 1-183) and a C-terminal domain (CTD; residues 184-387) (Figure 2a). The NTD contains a Rossmannlike α/β fold, in which a six-stranded parallel β-sheet (β1-β8-β5-β4-β2-β3) is centrally located and surrounded by six α-helices (α1-α6). One face of the Rossmann-like fold is also tion coefficient between intensities from random half-data sets. e R work = Σ||F obs |-|F calc ||/Σ|F obs | where F calc and F obs are the calculated and observed structure factor amplitudes, respectively. f R free = as described for R work , except that 5% of the total reflections were selected at random and omitted from refinement. g Calculated using MolProbity (http://molprobity.biochem.duke.edu, accessed on 11 December 2021).
YugJ Ni consists of two domains, an N-terminal domain (NTD; residues 1-183) and a C-terminal domain (CTD; residues 184-387) (Figure 2a). The NTD contains a Rossmannlike α/β fold, in which a six-stranded parallel β-sheet (β1-β8-β5-β4-β2-β3) is centrally located and surrounded by six α-helices (α1-α6). One face of the Rossmann-like fold is also decorated by the β6-β7 hairpin that protrudes from the β5 and β8 strands. The β1 strand is the most extended β-strand consisting of 11 residues and is antiparallelly aligned with its counterpart β-strand from the facing subunit, allowing YugJ to form a 12-stranded β-sheet and dimerize ( Figure 2b). In contrast to the NTD, the CTD exclusively consists of α-helices and can be divided into two α-helix bundles (α7-α9 helices and α10-α15 helices). The NTD and CTD are organized to generate a cleft, which accommodates the NADP(H) cofactor (see below).
YugJ shows structural similarity to other group III AAORs, including E. coli YqhD (PDB ID 1OJ7), Klebsiella pneumoniae 1,3-propanediol dehydrogenase (PDB ID 3BFJ), and Zymomonas mobilis alcohol dehydrogenase 2 (PDB ID 3OWO) (Supplementary Figures S3 and S4) [12,42,43]. These group III AAORs exhibit significant sequence (sequence identity, 27%~37%) and structure (root-mean-square deviation, 1.7~2.3 Å) similarities to YugJ, indicating that YugJ is a group III AAOR. In contrast to the overall structural similarity of group III AAORs, the local structures of the α9-α10 loop are categorized into two conformations depending on length (Supplementary Figure S5). The α9-α10 loops of most group III AAORs are short and make a tight turn between the α9 and α10 helices. However, the α9-α10 loop of YugJ is longer and adopts a more extended coil structure like that of E. coli YqhD. Interestingly, this unique structure of the YugJ α9-α10 loop is involved in regulation of access to the active site (see below).
In the YugJ Ni structure, a strong electron density peak that belongs to a metal ion was found in each YugJ monomer, although a metal ion was not used for crystallization. To define the identity of the metal ion, an X-ray fluorescence spectrum was obtained from a YugJ crystal [44]. In the spectrum, a prominent peak was observed at 7.46 keV, which corresponds to the X-ray emission energy of the Ni 2+ ion, indicating that the metal ion observed in the YugJ structure is a Ni 2+ ion (Figure 2c). The Ni 2+ ion is located between the two α-helix bundles of the CTD in the interdomain cleft and coordinated by conserved aspartate (Asp194) and histidine (His198, His267, and His281) residues (Figure 2a). Given that YugJ exhibited high aldehyde reductase activity in the presence of Ni 2+ ions and was observed with Ni 2+ ions in the crystal structure of YugJ Ni , we conclude that YugJ is an atypical group III AAOR that favors a Ni 2+ ion as a metallic cofactor.

NADP(H) Recognition by YugJ
Group III AAORs use NADPH or NADH as a reducing cofactor for catalysis. To define the cofactor specificity of YugJ, the HMF-reducing activity of YugJ was measured in the presence of NADPH or NADH. YugJ was highly active as an HMF reductase when supplemented with NADPH, but it was inactive with NADH, indicating that YugJ is an NADPH-dependent aldehyde reductase (Figure 3a).
To address the specific interaction of YugJ with NADP(H), we determined the crystal structures of YugJ in complex with NADP in two crystal forms (Supplementary Figure S6 and Table 1). The first crystal structure (space group P1) contains both the NADP cofactor and Ni 2+ ion and was named YugJ NADP-Ni . The second NADP-bound structure (space group P2 1 ) additionally harbors nitrate ions near NADP and was named YugJ NADP-NO3 . The YugJ NADP-NO3 structure exhibits well-defined electron density for each atom of NADP. However, in the YugJ NADP-Ni structure, the nicotinamide ring of NADP is disordered, and only the remaining NADP region was built (Supplementary Figure S6). Therefore, the interaction between YugJ and NADP is described with the YugJ NADP-NO3 structure unless otherwise specified.
In the YugJ NADP-NO3 structure, the NADP molecule is deeply embedded into the cleft between the two domains of YugJ (Figure 3b). NADP leans toward the NTD in the interdomain cleft and exhibits more interactions with the NTD than with the CTD. Each YugJ polypeptide chain from the dimer binds one NADP molecule with a buried surface area of~690 Å 2 , mainly via hydrogen bonds and van der Waals interactions. The adenine moiety of NADP is sandwiched between the Ser41 and Val183 residues and forms hydrogen bonds with YugJ residues from the β5-β6 loop (Thr138) and β8-α7 loop (Asn179 and Thr182). The pyrophosphate group in the middle of NADP is located in close proximity to the conserved cofactor-binding motif of aldehyde reductase enzymes (Gly96, Gly97, Gly98, and Ser99 residues; GGGS 1 motif; Supplementary Figure S3) and is stabilized via hydrogen bonds with the Gly98 and Ser99 residues from the α5 helix. The nicotinamide ring and its linked ribose moiety in NADP also form multiple hydrogen bonds with YugJ residues. The YugJ Asn71 and Lys160 residues at the β3-α4 loop and β7 strand, respectively, interact with the hydroxyl groups of the NADP ribose moiety. The nicotinamide ring interacts with the YugJ Asp102, Asn147, and Gly149 residues from the α5 helix, β5-β6 loop, and β6 strand, respectively, in the YugJ NADP-NO3 structure. However, in the YugJ NADP-Ni structure, the nicotinamide-YugJ interaction is not observed because of nicotinamide disorder.
corresponds to the X-ray emission energy of the Ni 2+ ion, indicating that the metal ion observed in the YugJ structure is a Ni 2+ ion (Figure 2c). The Ni 2+ ion is located between the two α-helix bundles of the CTD in the interdomain cleft and coordinated by conserved aspartate (Asp194) and histidine (His198, His267, and His281) residues (Figure 2a). Given that YugJ exhibited high aldehyde reductase activity in the presence of Ni 2+ ions and was observed with Ni 2+ ions in the crystal structure of YugJ Ni , we conclude that YugJ is an atypical group III AAOR that favors a Ni 2+ ion as a metallic cofactor.

NADP(H) Recognition by YugJ
Group III AAORs use NADPH or NADH as a reducing cofactor for catalysis. To define the cofactor specificity of YugJ, the HMF-reducing activity of YugJ was measured in the presence of NADPH or NADH. YugJ was highly active as an HMF reductase when supplemented with NADPH, but it was inactive with NADH, indicating that YugJ is an NADPH-dependent aldehyde reductase (Figure 3a).  The NADP cofactor (carbon, green sphere; nitrogen, blue sphere; oxygen, red sphere; phosphorus, orange sphere; interatomic bond, green stick) and its neighboring nitrate ion (nitrogen, blue sphere; oxygen, red sphere; interatomic bond, yellow stick) are represented by ball-and-stick models. The NADP-binding residues of YugJ are shown as gray lines on the YugJ structure (gray ribbons). In particular, the YugJ residues that form polar interactions with NADP are depicted as gray sticks. The YugJ-NADP complex structure allows us to explain why YugJ prefers NADPH to NADH as a reducing cofactor. At the 2 position of the adenine-linked ribose moi-ety, NADP(H) has an additional phosphate group that is not present in NAD(H). In the YugJ NADP-NO3 structure, the 2 -phosphate group of NADP is inserted into the curved structure of the second GGGS motif (GGGS 2 motif; Gly38-Ser41 residues) at the β2-α2 loop (Figure 3b). The GGGS 2 motif extensively interacts with the 2 -phosphate group of NADP using the Gly39-Ser41 main chains and the Ser41 side chain. The GGGS 2 motif is highly conserved across NADP(H)-dependent group III AAOR enzymes (YqhD in Supplementary Figure S3) [12]. However, in NAD(H)-dependent group III AAOR enzymes, the Gly38 residue at the GGGS 2 motif is substituted with an aspartate residue, which sterically clashes with the 2 -phosphate group of NADP(H) (K. pneumoniae 1,3-propanediol dehydrogenase and Z. mobilis alcohol dehydrogenase 2 in Supplementary Figure S3) [42]. Overall, we conclude that the conserved GGGS 2 motif of YugJ plays a key role in distinguishing NADP(H) from NAD(H).
A comparative analysis of the NADP-free and NADP-bound YugJ structures revealed an NADP binding-mediated change in the interdomain dynamics of YugJ (Figure 3c). YugJ exhibits interdomain flexibility in the absence of NADP, given that YugJ chains A and B in the YugJ Ni structure adopt open and closed conformations, respectively, which differ in interdomain angle by~10 • (Supplementary Figure S7a). However, NADP binding restricts the interdomain organization of YugJ into the closed conformation. Each protomer of the NADP-bound YugJ structures (YugJ NADP-Ni chains A, B, C, and D and YugJ NADP-NO3 chains A and B) adopts a closed conformation with a smaller interdomain angle, similar to that of YugJ Ni chain B (Figure 3c and Supplementary Figure S7b). Thus, the YugJ structures in complex with NADP form more optimized interactions with NADP and Ni 2+ in a narrower interdomain cleft than the NADP-free structures. Based on these findings, we propose that YugJ shifts its dynamic equilibrium toward a closed conformation upon NADPH binding.

Putative Substrate-Binding Site of YugJ
Because YugJ accommodates NADP and metal cofactors in the interdomain cleft, the cleft highly likely functions as an active site that binds and transforms the substrate. To provide insights into the substrate-binding site of YugJ, we carefully analyzed the electron density of the YugJ structures in the interdomain cleft. Notably, in the YugJ NADP-NO3 crystal structure, a three-pointed star-like electron density was observed near the nicotinamide ring of NADP in the interdomain cleft (Figure 4a). The extra density is most likely derived from a nitrate ion, given that nitrate ions were used for YugJ NADP-NO3 crystallization and resemble a three-pointed star. Accordingly, an additional three-pointed density was not observed in the YugJ Ni or YugJ NADP-Ni structure obtained in the absence of nitrate. Therefore, the three-pointed star-like electron density was modeled as a nitrate ion in the YugJ NADP-NO3 structure.
In the YugJ NADP-NO3 structure, the nitrate ion is enclosed by NADP and YugJ residues from the α9-α10 loop and α10 and α11 helices. The nitrate ion forms hydrogen bonds with the nicotinamide-linked ribose moiety of NADP and the imidazole ring of the YugJ His271 residue. Noticeably, the oxygen atom of the nitrate ion is located in close proximity to the C-4 atom of the nicotinamide moiety that undergoes oxidation upon substrate reduction, suggesting that the oxygen atom of the nitrate ion positionally mimics that of the aldehyde substrate. Interestingly, other aldehyde reductase structures have been reported to accommodate various chemicals at or near the nitrate-binding site of the YugJ NADP-NO3 structure [12,14,45]. Based on these observations, we propose that the nitrate-binding site is used by YugJ to recognize the aldehyde substrate. To confirm this proposal, a competitive inhibition assay was performed using nitrate (Figure 4b). The HMF-reducing activity of YugJ decreased by~75% in the presence of lithium nitrate, suggesting that nitrate ions compete with HMF to bind the active site of YugJ. The inhibitory effect seems to be specific to nitrate, given that lithium sulfate addition did not significantly reduce YugJ activity. structure, a three-pointed star-like electron density was observed near the nicotinamide ring of NADP in the interdomain cleft (Figure 4a). The extra density is most likely derived from a nitrate ion, given that nitrate ions were used for YugJ NADP-NO3 crystallization and resemble a three-pointed star. Accordingly, an additional three-pointed density was not observed in the YugJ Ni or YugJ NADP-Ni structure obtained in the absence of nitrate. Therefore, the three-pointed star-like electron density was modeled as a nitrate ion in the YugJ NADP-NO3 structure. Noticeably, YugJ exhibits substantial structural differences at the α9-α10 loop, which neighbors the nitrate ion in the YugJ NADP-NO3 structure (Figure 4c). YugJ NADP-NO3 locates the α9-α10 loop closer to the active site than the YugJ Ni and YugJ NADP-Ni structures and forms an additional α-helix in the middle of the α9-α10 loop. These structural changes seem to be induced by the YugJ NADP-NO3 -specific interactions of the α9-α10 loop residues, Trp264 and Arg261, with the nitrate ion and β5-β6 loop, respectively. As a result of the structural rearrangement, the α9-α10 loop completely covers the nitrate ion in the YugJ NADP-NO3 structure and induces closure of the active site. In contrast, the α9-α10 loop of the YugJ Ni and YugJ NADP-Ni structures adopts an open conformation that increases the accessibly of the active site. Given that nitrate inhibits the HMF-reducing activity of YugJ presumably via a competitive inhibition mechanism, we propose that the α9-α10 loop functions as a lid that closes the active site upon substrate binding for substrate stabilization.
Nitrate binding appears to induce structural changes in NADP(H) in addition to the YugJ α9-α10 loop. In the YugJ NADP-NO3 structure, the nicotinamide ring of NADP adopts a rigid structure, interacting with the nitrate ion (Figure 4a and Supplementary Figure S6a). On the other hand, in the nitrate-less YugJ NADP-Ni structure, the nicotinamide ring of NADP is disordered, although similar conformations are observed for the YugJ Asp102, Asn147, and Gly149 residues that interact with nicotinamide in the YugJ NADP-NO3 structure (Figure 3b and Supplementary Figure S6b). Thus, we propose that NADP(H) nicotinamide in YugJ prefers high structural dynamics for substrate recognition and becomes rigid upon substrate binding.
To further investigate the substrate-binding mechanism of YugJ, we modeled the HMF-bound YugJ structure through in silico molecular docking [46]. In the complex model, HMF is found near the nicotinamide moiety of NADP in the interdomain cleft, and the aldehyde group of HMF is oriented toward the C-4 atom of the nicotinamide ring and the Ni 2+ ion ( Figure 5). Notably, the aldehyde group of HMF sterically clashes with the nitrate ion from the overlaid YugJ NADP-NO3 structure (Supplementary Figure S8). In the YugJ-HMF model, YugJ residues clasp and stabilize the termini of HMF (aldehyde and hydroxyl groups), properly orienting HMF for reduction. The His271 residue from the CTD α10 helix forms a hydrogen bond with the aldehyde group of HMF. The contribution of His271 to substrate binding has been reported in previous mutation studies on an E. coli homolog of YugJ [9,47]. The Ser150 and Trp163 residues from the NTD β6 and β7 strands, respectively, directly interact with the hydroxyl group of HMF, explaining why furfural, which lacks the hydroxyl group, is less efficiently reduced by YugJ than HMF. of His271 to substrate binding has been reported in previous mutation studies on an E. coli homolog of YugJ [9,47]. The Ser150 and Trp163 residues from the NTD β6 and β7 strands, respectively, directly interact with the hydroxyl group of HMF, explaining why furfural, which lacks the hydroxyl group, is less efficiently reduced by YugJ than HMF. Figure 5. YugJ-HMF complex model based on molecular docking. HMF was docked on the YugJ-NADP-Ni 2+ model. HMF and NADP are represented by light blue and green ball-and-stick models, respectively. The Ni 2+ ion is represented by a cyan sphere. The HMF-binding residues and Ni 2+coordinating residues of YugJ are shown as gray sticks on the YugJ structure (gray ribbons). The hydrogen bonds of HMF with YugJ residues are represented by black dotted lines. The aldehyde and hydroxyl groups of HMF are indicated by blue and red dotted boxes, respectively.
In conclusion, we identified B. subtilis YugJ as an NADPH-dependent furan aldehyde reductase based on structural and biochemical studies. Our comparative analysis of the YugJ structures allows us to propose initial catalytic events that occur during YugJ-mediated HMF reduction ( Figure 6). First, NADPH is inserted into the interdomain cleft of YugJ, limiting the interdomain dynamics of YugJ into a closed conformation. Next, the HMF substrate binds the active site near the NADPH and metal cofactors and is stabilized via closure of the α9-α10 loop lid for subsequent product formation. Our findings and proposal will provide a structural basis for understanding the aldehyde reduction mechanism of YugJ and designing enzymes with improved furan aldehyde reduction activity. YugJ-HMF complex model based on molecular docking. HMF was docked on the YugJ-NADP-Ni 2+ model. HMF and NADP are represented by light blue and green ball-and-stick models, respectively. The Ni 2+ ion is represented by a cyan sphere. The HMF-binding residues and Ni 2+coordinating residues of YugJ are shown as gray sticks on the YugJ structure (gray ribbons). The hydrogen bonds of HMF with YugJ residues are represented by black dotted lines. The aldehyde and hydroxyl groups of HMF are indicated by blue and red dotted boxes, respectively.
In conclusion, we identified B. subtilis YugJ as an NADPH-dependent furan aldehyde reductase based on structural and biochemical studies. Our comparative analysis of the YugJ structures allows us to propose initial catalytic events that occur during YugJ-mediated HMF reduction ( Figure 6). First, NADPH is inserted into the interdomain cleft of YugJ, limiting the interdomain dynamics of YugJ into a closed conformation. Next, the HMF substrate binds the active site near the NADPH and metal cofactors and is stabilized via closure of the α9-α10 loop lid for subsequent product formation. Our findings and proposal will provide a structural basis for understanding the aldehyde reduction mechanism of YugJ and designing enzymes with improved furan aldehyde reduction activity.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 12 of 17 Figure 6. A model for YugJ binding to NADPH and HMF. The YugJ NTD and CTD are shown as blue and red ribbons, respectively, and the α9-α10 loop lid in the CTD is highlighted in yellow. The NADPH and Ni 2+ cofactors are depicted as green sticks and a cyan sphere, respectively. NADPH and HMF binding-induced structural changes in YugJ are represented by thick red arrows.

Construction of a YugJ Expression Plasmid
The open reading frame that encodes the full-length YugJ protein (GenBank: EFG92987.1; residues 1-387) was amplified by PCR from the genomic DNA of B. subtilis subsp. spizizenii using primers containing the BamHI or SalI recognition sequence. The PCR product was digested using the BamHI and SalI restriction enzymes and ligated into a pET49b plasmid that had been modified to express the recombinant protein in fusion with a His6 tag and a thrombin cleavage site at the N-terminus. The ligation product was transformed into E. coli DH5α cells. The nucleotide sequence of the YugJ gene in the pET49b plasmid was verified by DNA sequencing.

Expression and Purification of the YugJ Protein
The YugJ expression plasmid was transformed into E. coli BL21 (DE3) cells for YugJ overexpression. Transformant cells were grown at 37 °C in LB medium until the optical density at 600 nm reached 0.6-0.8. The YugJ protein was overexpressed in the presence of 1 mM isopropyl-β-D-1-thiogalactopyranoside at 18 °C overnight. The E. coli cells were harvested by centrifugation and lysed by sonication in a solution containing 50 mM Tris, pH 8.0, 200 mM NaCl, 5 mM β-mercaptoethanol, and 1 mM phenylmethanesulfonyl fluoride. The cell lysate was cleared by centrifugation, and the resultant supernatant was incubated with Ni-NTA resin (Qiagen, Venlo, Netherlands) to purify the His6-tagged YugJ Figure 6. A model for YugJ binding to NADPH and HMF. The YugJ NTD and CTD are shown as blue and red ribbons, respectively, and the α9-α10 loop lid in the CTD is highlighted in yellow. The NADPH and Ni 2+ cofactors are depicted as green sticks and a cyan sphere, respectively. NADPH and HMF binding-induced structural changes in YugJ are represented by thick red arrows.

Construction of a YugJ Expression Plasmid
The open reading frame that encodes the full-length YugJ protein (GenBank: EFG92987.1; residues 1-387) was amplified by PCR from the genomic DNA of B. subtilis subsp. spizizenii using primers containing the BamHI or SalI recognition sequence. The PCR product was digested using the BamHI and SalI restriction enzymes and ligated into a pET49b plasmid that had been modified to express the recombinant protein in fusion with a His 6 tag and a thrombin cleavage site at the N-terminus. The ligation product was transformed into E. coli DH5α cells. The nucleotide sequence of the YugJ gene in the pET49b plasmid was verified by DNA sequencing.

Expression and Purification of the YugJ Protein
The YugJ expression plasmid was transformed into E. coli BL21 (DE3) cells for YugJ overexpression. Transformant cells were grown at 37 • C in LB medium until the optical density at 600 nm reached 0.6-0.8. The YugJ protein was overexpressed in the presence of 1 mM isopropyl-β-D-1-thiogalactopyranoside at 18 • C overnight. The E. coli cells were harvested by centrifugation and lysed by sonication in a solution containing 50 mM Tris, pH 8.0, 200 mM NaCl, 5 mM β-mercaptoethanol, and 1 mM phenylmethanesulfonyl fluoride. The cell lysate was cleared by centrifugation, and the resultant supernatant was incubated with Ni-NTA resin (Qiagen, Venlo, Netherlands) to purify the His 6 -tagged YugJ protein via affinity chromatography. The YugJ protein was eluted from Ni-NTA resin using 250 mM imidazole and dialyzed against 20 mM Tris, pH 8.0, and 5 mM βmercaptoethanol. The resulting protein was incubated with thrombin at 18 • C for 24 h to remove the N-terminal His 6 tag. The tag-free YugJ protein was loaded onto a Mono Q 10/100 GL column (GE Healthcare, Chicago, IL, USA) for further purification by anionexchange chromatography and eluted from the Mono Q resin using a 0-500 mM NaCl gradient. The oligomeric state of the purified YugJ protein was analyzed by gel-filtration chromatography using a Superdex 200 10/300 column (GE Healthcare, Chicago, IL, USA) in 20 mM Tris, pH 8.0, 150 mM NaCl, and 5 mM β-mercaptoethanol.

Crystallization and X-ray Diffraction
YugJ crystallization conditions were screened with JCSG Core Suites (Qiagen, Venlo, Netherlands) and optimized by the sitting-drop vapor-diffusion method at 18 • C using YugJ protein in 20 mM Tris, pH 8.0, 200 mM NaCl, and 5 mM β-mercaptoethanol. YugJ Ni crystals were generated by mixing 0.5 µL of YugJ protein (12.7 mg/mL) with 0.5 µL of a well solution containing 20% PEG 6000 and 0.1 M Tris, pH 8.0, and then equilibrating the resultant 1-µL drop against 500 µL of the well solution. To obtain YugJ NADP-NO3 crystals, YugJ protein (18.3 mg/mL) was mixed with NADP at a molar ratio of 1:3, and the resulting mixture was equilibrated to a well solution containing 18% PEG 3350, 0.1 M MES, pH 6.5, and 0.3 M ammonium nitrate. To generate YugJ NADP-Ni crystals, YugJ crystals were first obtained in a drop containing 0.5 µL of YugJ protein (13.6 mg/mL) and 0.5 µL of a well solution (14% PEG 8000, 0.2 M calcium acetate, and 0.1 M MES, pH 6.5) and were then soaked in a solution containing 3 mM NADP and 3 mM HMF.
A YugJ crystal was briefly soaked in a cryoprotectant solution containing 25% glycerol or 25% ethylene glycol and flash-cooled under a nitrogen cryostream. X-ray diffraction was performed at the Pohang Accelerator Laboratory (Pohang, Korea), and diffraction data were reduced and scaled using the HKL2000 package [48]. To verify the presence of Ni 2+ ions in the YugJ crystal, the X-ray fluorescence emission spectrum was obtained from the YugJ crystal. Data collection statistics are summarized in Table 1.

Structure Determination
The YugJ Ni structure was determined by molecular replacement with the Phaser program using the structure of T. maritima butanol dehydrogenase A (PDB ID 1VLJ) as a search model [49]. Model building and refinement were performed using the Coot and Phenix programs, respectively, to yield the final structure of YugJ Ni [50,51]. The YugJ NADP-NO3 and YugJ NADP-Ni structures were solved by molecular replacement using the YugJ Ni structure as a search model. The final YugJ NADP-Ni and YugJ NADP-NO3 structures were obtained via iterative cycles of model building and refinement [50,51]. The atomic coordinates and the structure factors for the YugJ Ni , YugJ NADP-Ni , and YugJ NADP-NO3 structures (PDB ID 7W9X, 7W9Y, and 7W9Z, respectively) have been deposited in the Protein Data Bank.
Structure refinement statistics indicate that the three structures are overall of high quality (Table 1). However, the YugJ NADP-Ni structure has two Ramachandran outliers at the Ala141 residues from chains B and D. The two YugJ NADP-Ni residues are located at the boundary between the favored region and outlier region of the Ramachandran plot, as observed for the Ala141 residues from chains A and C. Moreover, these four residues exhibit similar conformations. Notably, the YugJ Ni structure displays a higher average B-factor (48.7 Å 2 ) than the YugJ NADP-Ni (29.4 Å 2 ) and YugJ NADP-NO3 (22.2 Å 2 ) structures, presumably due to lower resolution and a more disordered crystal lattice. However, the quality of the data collected for YugJ Ni is similar to those of YugJ NADP-Ni and YugJ NADP-NO3 .

Measurement of the Catalytic Activity of YugJ
The enzymatic activity of YugJ was evaluated using demetallized YugJ protein. To obtain metal-free YugJ protein, the purified recombinant YugJ protein was treated with a solution containing 1 mM EDTA and 100 mM sodium acetate, pH 5.0, at 4 • C for 2 h and subsequently diluted~90-fold using a solution containing 20 mM Tris, pH 8.0, and 150 mM NaCl [52]. The metal-free YugJ protein (12 µM) was preincubated with divalent metal ions (120 µM) prior to the measurement of catalytic activity.

In Silico Molecular Docking Analysis
The structure of YugJ in complex with HMF was modeled via in silico molecular docking. The coordinates of HMF were obtained in the SDF format from the PubChem database [53]. The coordinate files of the HMF ligand and YugJ protein were converted to the PDBQT format using the AutoDock Tools package. Next, the HMF ligand was docked on the YugJ protein using the AutoDock Vina program [46]. The docked ligands were analyzed based on binding energy. The YugJ-HMF complex model was graphically represented using the PyMOL program.

Data Availability Statement:
The atomic coordinates and the structure factors for YugJ (PDB ID 7W9X, 7W9Y, and 7W9Z) have been deposited in the Protein Data Bank (www.pdb.org, deposited on 11 December 2021).